Penicillin-binding proteins (PBPs) play an essential role in the synthesis and maintenance of the bacterial cell wall and are the molecular targets of the widely used ?-lactam antibiotics. In the face of the increasingly urgent threat of antibacterial resistance, PBPs present new opportunities for novel antibiotic development, particularly against multi-drug resistant pathogens such as Pseudomonas aeruginosa. This proposal focuses on P. aeruginosa PBP3, which is an essential enzyme in P. aeruginosa. We aim to develop non-?-lactam inhibitors that will not be susceptible to existing resistance mechanisms of ?-lactamases, by utilizing the scaffolds of two latest covalent ?-lactamase inhibitors, diazabicyclooctane (DBO) and cyclic boronate (CB) compounds, and by developing novel non-covalent inhibitors. DBO and CB inhibitors react with the catalytic serine of ?-lactamases, and can form the same covalent adducts with PBPs, which share many key catalytic residues and active site features with serine ?-lactamases. Although most existing DBO and CB compounds are not potent PBP inhibitors, we hypothesize that we can promote covalent adduct formation by enhancing the non-covalent interactions between these compounds and PBPs, thus stabilizing and orienting the inhibitor for the chemical reaction. Through structure-based design and synthesis, we will optimize non-covalent interactions between DBO/CB compounds and PBP3. We will analyze both the overall reactivity of the covalent inhibitors (i.e., formation of the covalent adduct in biochemical experiments) and the non-covalent binding (assayed by surface plasmon resonance using a mutant lacking the catalytic serine). By obtaining a quantitative and detailed understanding of the effects of the non-covalent interactions on inhibitor activity, we hope to engineer DBO/CB compounds ultimately as broad-spectrum inhibitors, while developing SPR as a platform to analyze and screen PBP inhibitors. Additionally, we will apply virtual screening to identify novel non-covalent inhibitors, an approach that we have previously successfully employed to develop ?-lactamase inhibitors. These studies will lead to new chemical matter for future antibiotic discovery efforts against P. aeruginosa, while offering important insights into novel inhibitor discovery targeting PBPs in other bacteria.